Signals through the TCR and other surface receptors determine whether thymocytes and mature T cells will grow, survive, differentiate, and die. Our previous studies and those of others indicated that the strength and/or duration of TCR signaling influence lineage decisions. We have collaborated with Remy Bosselut's lab to better understand how two transcription factors, ThPOK and GATA3, operate in CD4/CD8 T lineage commitment. It was established that both of these factors are required for CD4 T cell development, but only ThPOK appeared to play a pivotal role in the lineage decision. The failure to find any perturbation in TCR signaling pathways or in lineage commitment in conditionally GATA3-deficient mice led others to the proposal that GATA3 was critical in the maturation of CD4+ thymocytes, after lineage commitment. Nevertheless, previous results from our lab indicated that re-directed lineage choice, as a result of attenuated TCR signaling, could only be observed when MHC recognition was mediated by a coreceptor-independent, high affinity TCR. To revisit this question, we generated GATA3-deficient mice with only the MHC2-restricted 5C.C7 TCR. Although control mice generated large numbers of CD4+ thymocytes as expected, predominantly CD8 thymocytes developed in the absence of GATA3, providing evidence that cell fate was re-directed in the mutant mice. Similarly, when GATA3-deficient bone marrow was used to reconstitute irradiated MHC1-deficient recipients, very few mature CD4 thymocytes appeared, but substantial numbers of MHC2-selected CD8 thymocytes were generated (Nat Immunol 9:1122). These results suggested an important role GATA3 during or developmentally upstream of CD4/CD8 lineage commitment. From these data, it was conceivable that the function of GATA3 was to induce expression of ThPOK, the key factor specifying CD4 T cell fate. Indeed, we showed in the same study that GATA3 was recruited to the ThPOK locus and required to trigger ThPOK expression. However, introduction of a ThPOK transgene into GATA3-deficient mice failed to rescue CD4 T cell development, indicating that GATA3 was not the only essential factor critical for ThPOK expression and/or that GATA3 was indispensible for earlier stages of positive selection necessary for ThPOK induction. In fact, it was already established that GATA3 expression could be upregulated by TCR signaling in positive selection and that ThPOK was not induced until a mid to late stage of CD4 T cell development. Collectively, these results suggested that GATA3 may act as a triggering or specification factor, while ThPOK serves as an enforcement factor for CD4 T cell commitment. The implication that GATA3 was needed at early stages of positive selection could explain why it had been difficult to demonstrate its contribution to lineage commitment. That is, most thymocytes would not progress to the stage at which lineage re-direction could be visualized in the absence of GATA3. To investigate Notch functions in development, we have generated Presenilin(PS) conditionally mutant mice in which all Notch activity can be prohibited in specific tissues. With gene deletion in the thymus, the generation of CD4 SP thymocytes was inefficient with a diverse TCR repertoire and severely compromised in mice expressing a single MHC2-restricted TCR. Diminished T cell production correlated with impaired TCR signaling in PS-deficient thymocyte precursors that could be rescued with an activated form of Notch. Our evidence for a functional link between Notch and TCR signaling pathways in developing thymocytes led us to examine whether these pathways were similarly associated in naive CD4 T cells responding to antigen. Previous reports suggested that Notch signaling controls T helper (Th1/Th2) differentiation of naive CD4 T cells, although the mechanism responsible for this regulation was controversial. Analogous to the model for thymic development in which the quantity of TCR signal biases the CD4/CD8 cell fate decision, there is a strength of signal model for Th1 /Th2 lineage commitment in mature T cell responses, where low doses of antigenic peptide promote the Th2 fate, and high doses of peptide promote the Th1 fate. To determine the role of Notch in T helper differentiation, we engineered a new mouse model with deletion of PS genes targeted to peripheral, naive T cells bearing a single 5C.C7 TCR. In collaboration with William Paul's lab, we used an in vitro system in which purified naive CD4 T cells were stimulated with low doses of specific peptide presented by antigen presenting cells (APC) under non-polarizing conditions. Control T cells proliferated well and made IL-4 in this system, while PS-deficient cells proliferated poorly and failed to differentiate into IL4-secreting Th2 lineage cells. Previous studies had used only a single targeted mutation, making it impossible to determine the mechanism(s) by which Notch proteins influence Th2 differentiation. Therefore, we generated two additional lines bearing the 5C.C7 TCR, one with targeted deletion of Pofut1 (an O-fucosyl-transferase1 enzyme essential for the surface expression of Notch receptors) and one with targeted deletion of CSL (a DNA-binding protein involved in regulating transcription of Notch target genes). By comparing phenotypes of these Notch mutants, we could distinguish CSL-dependent versus CSL-independent mechanisms. Notably, CSL- and Pofut1-deficient mice, similar to the PS-deficient mice, proliferated poorly and failed to secrete IL-4 in response to low dose peptide stimulation. In all three mutants, defects in IL-4 production could be rescued by addition of exogenous IL-2, suggesting IL-2 production to be the major defect early in the response. Moreover, the defect in IL2 production was correlated with less nuclear translocation of NFATc2. Collectively, these studies indicate that Notch indirectly influences Th2 differentiation by controlling optimal IL-2 production. To investigate whether these vitro findings have in vivo and clinical significance, we initiated collaborative studies of Schistosomiasis with Thomas Wynn's laboratory. Schistosomiasis is a major parasitic disease in humans. The mouse model for the disease develops granulomas and fibrosis, similar to those seen in human schistosome infection. This is an ideal model system to investigate the role of Notch in CD4 T helper cell differentiation, because S. mansoni egg antigens are extremely potent inducers of Th2 responses. During a natural infection, mature Schistosome worms lay eggs in the portal vein, and eggs that accumulate in the lungs lead to pulmonary fibrosis. We find that control mice given a single intravenous injection of eggs synchronously develop granuloma formation around the eggs in the lungs. However, mice with PS-, CSL- or Pofut1-deficient T cells, showed reduced lymph node cellularity, fewer T cells secreting Th2 cytokines (IL-4, IL-5, IL-13), decreased pulmonary fibrosis, and decreased levels of serum IgE. These results indicate that Notch signaling in T cells is critical for normal immune responses to helminth infection in vivo. Of importance, the in vitro and in vivo experiments collectively support a model in which the primary defect resulting from Notch inhibition is in T cell activation. Thus, similar to developing thymocytes, there is a functional link between Notch and TCR signaling in T helper differentiation in peripheral CD4 T cells.